Heterocyclic compounds, method of developing new drug leads and combinatorial libraries used in such method

ABSTRACT

The present invention provides according to a first of its aspects, new compounds that have a flexible scaffold with various degrees of conformational restriction and accordingly are useful as drug candidates. These compounds may be used to produce new combinatorial libraries that will permit to screen for and select drug candidates for a variety of uses in human medicine, veterinary medicine and in agriculture.

FIELD OF THE INVENTION

[0001] This invention is in the field of using combinatorial chemistryto develop new drugs.

LIST OF REFERENCES

[0002] The following references are considered to be pertinent for thepurpose of understanding the background of the present invention:

[0003] Adang A. E. P. and Hermkens P. H. H., Curr. Med. Chem. 8:985(2001);

[0004] Beeley N. R. A., Drug Disc. Today 5:354 (2000);

[0005] Bunin B. A. and Ellman J. A., J. Am. Chem. Soc. 114:11997 (1992);

[0006] Campian E. et al, Bioorg. Med. Chem Lett. 8;2357(1998);

[0007] Furka A. et al. Int. J. Pept. Protein. Res. 37:487-493 (1991);

[0008] Geysen H. M. et al. Proc. Natl. USA, 81: 3998 (1984);

[0009] Hougten R. A., Proc. Natl. USA, 82:5131 (1985);

[0010] Kumar S. et al. Prot. Sci. 9:10-19 (2000);

[0011] Lipinsky C. A. et al., Adv. Drug Deliv. Rev. 23, 3 (1997);

[0012] Lipinsky C. A., Chimia 52:503 (1998);

[0013] Morrison K. L. and Weiss G. A., Curr. Opin. Chem. Biol. 5:302-307(2001);

[0014] Rink H., Tetrahedron Lett, 28: 3787 (1987);

[0015] Winter et al. Nature 299: 756 (1982);

[0016] The above references will be acknowledged in the text below byindicating the author's name and year of publication (in brackets) fromthe above list.

BACKGROUND OF THE INVENTION

[0017] An important objective of combinatorial chemistry is to generatea large number of novel compounds that can be screened to identify leadcompounds for pharmaceutical research. Theoretically, the total numberof compounds which may be produced for a given library is limited onlyby the number of reagents available to form substituents on the variablepositions on the library's molecular scaffold.

[0018] The combinatorial process lends itself to automation, both in thegeneration of compounds and in their biological screening, therebygreatly enhancing the opportunity and efficiency of drug discovery.Combinatorial chemistry may be performed in a manner where libraries ofcompounds are generated as mixtures, while the complete identificationof the individual compounds is postponed until after positive screeningresults are obtained. However, a preferred form of combinatorialchemistry is “parallel array synthesis”, (also called Multiple ParallelSynthesis, MPS) where individual reaction products are simultaneouslysynthesized, but are retained in separate compartments. [Geysen et al.(1984); Hougten (1985)]. For example, the individual library compoundscan be prepared, stored, and assayed in separate wells of a microtiterplate, each well containing one member of the parallel array, The use ofstandardized microtiter plates or equivalent apparatus, is advantageousbecause such an apparatus is readily accessed by programmed roboticmachinery, both during library synthesis and during library sampling orassaying.

[0019] Combinatoral chemistry can be carried out in solution phase whereboth reactants are dissolved in solution or in solid phase where one ofthe reactants is covalently bound to a solid support. Solid phasesynthesis offers the advantage that reactions can be carried out usingexcess reagents, while the solid support-bound products are easilywashed free of excess reagent. The use of excess reagents may ensurehigh yield of each step in a multiple step synthesis. Solution phasesynthesis typically requires use of one or more reaction mixture work-upprocedures to separate reaction product from unreacted excess reagent.

[0020] The first combinatorial libraries were composed of peptides, inwhich all or selected amino acid positions were randomized [Geysen etal. (1984); Furka et al. (1991)].

[0021] Peptides and proteins can exhibit high and specific bindingactivity, and can act as catalysts. In consequence, they are of greatimportance in biological systems. Unfortunately, peptides per se havelimited utility for use as therapeutic entities. They are costly tosynthesize, unstable in the presence of proteases, non selective and ingeneral do not pass cellular membranes.

[0022] Nucleic acids have also been used in combinatorial libraries.Their great advantage is the ease with which a nucleic acid withappropriate binding activity can be amplified. As a result,combinatorial libraries composed of nucleic acids can be of lowredundancy and hence, of high diversity. However, the resultingoligonucleotides are not suitable as drugs for several reasons. First,the oligonucleotides have high molecular weights and cannot besynthesized conveniently in large quantities. Second, becauseoligonucleotides are polyanions, they do not cross cell membranes.Finally, deoxy- and ribo-nucleotides are hydrolytically digested bynucleases that occur in all living systems and are therefore usuallydecomposed before reaching the target.

[0023] There has therefore been much interest in combinatorial librariesbased on small molecules (i.e. molecules having molecular weight of upto about 1000 daltons), which are more suited to pharmaceutical use,especially those which, like benzodiazepines, belong to a chemical classwhich has already yielded useful pharmacological agents [Bunin andEllman (1992); Beeley (2000)]. The techniques of combinatorial chemistryhave been recognized as the most efficient means for finding smallmolecules that act on these targets. At present, small moleculecombinatorial chemistry involves the synthesis of either pooled ordiscrete molecules that present varying arrays of functionality on acommon scaffold. These compounds are grouped in libraries that are thenscreened against the target of interest either for binding or forinhibition of biological activity [Adang and Hermkens (2001)].

[0024] The elements of diversity of libraries of currently availablescaffold based compounds having the general structure (A) showed below,are based mainly on sequential or positional diversity namely the orderin which the various R groups are arranged around the ring and chemicaldiversity that can arise from alterations in the chemical nature of theR groups.

[0025] In the above structure (A), X, Y and Z represent ring heteroatomsor carbons, and R′, R″ and R′″ represent substituents associated to thering through a linker (showed schematically as a wavy line).

[0026] It is known from the art [Kumar S. et al. (2000)] that moleculesmay bind to each other if their conformations are complementary ingeometry and chemistry and if their binding produces stableassociations. However, most of the known libraries of organic moleculessuffer from a major drawback when applied for the discovery of new drugleads based on the inhibition of peptide: protein or protein: protein orprotein: nucleic acid interactions: they are too constrained andtherefore lack the ability to undergo conformational complementarity,i.e. lack an ability for binding to a protein and/or a nucleic acid.This led to the preparations of extremely large libraries (consist of upto millions of compounds) and their biological screening, which in manycases results in the discovery of low affinity leads or to the lack oftheir discovery.

SUMMARY OF THE INVENTION

[0027] The present invention provides, according to a first of itsaspects, new compounds that have a relatively flexible scaffold Thesecompounds may be used to produce new combinatorial libraries that willpermit, e.g. in high throughput screening assays, to screen for andselect drug candidates for a variety of uses in human medicine,veterinary medicine and in agriculture. The members of each libraryaccording to the invention differ from each other in the ring size, inaddition to the conventional chemical and positional diversity attainedby the different substituents on the scaffold, thus allowing theselection of the most active compound, not only on the basis of thenature and proper arrangement of the substituents (attained by the knownchemical and positional diversity), but also based on the ability toundergo conformational complementarity (attained by the conformationaldiversity).

[0028] Thus, the present invention provides scaffold based compoundshaving the following general formula (B):

[0029] including pharmaceutically acceptable salts, esters or solvatesthereof, wherein

[0030] Z is selected from C═O and —CH₂—,

[0031] W is selected from C═O and a bond, provided that at least one ofZ and W is C═O,

[0032] X and Y are independently selected from CH₂, O, S, NH, N—R⁵,NH—CO, CO, CH₂CO, S═O and SO₂, or X and Y may form together a groupselected from CH═CH, CO—NR⁵, NH—CO—NH, O—CH(R⁵)—O, NH—CH(R⁵)—O andNH—CH(R⁵)—NH, where the hydrogen in the above groups may optionally besubstituted by an alkyl group;

[0033] (a) and (b) are parts of the scaffold and are nitrogen containingbivalent organic radicals, each independently providing between 1 to 4,preferably up to 2 atoms to said scaffold,

[0034] R¹, R², R³ and R⁴ are each independently selected from H andsubstituted or unsubstituted alkyl, and

[0035] n and m are integers from 1 to 6.

[0036] Preferably, the present invention provides scaffold basedcompounds having the formula (B) above, wherein X and Y areindependently selected from CH₂, O, S, NH, N—R⁵, NH—CO, CO, CH₂CO, S═Oand SO₂, or X and Y may form is together a group selected from CO—NR⁵,NH—CO—NH, O—CH(R⁵)—O, NH—CH(R⁵)—O and NH—CH(R⁵)—NH, where the hydrogenin the above groups may optionally be substituted by an alkyl group.

[0037] According to a preferred embodiment, each of parts (a) and (b) isindependently selected from —N(CHR⁶CO—L)—, —C(CO—L)(R⁶)—,—N(COR⁸)—CHR⁷—, and —C(NHR⁸)(R⁷)—.

[0038] According to another preferred embodiment, the present inventionprovides heterocyclic compounds having the formula I, II, III or IV:

[0039] including pharmaceutically acceptable salts, esters or solvatesthereof, wherein

[0040] X and Y are independently selected from CH₂, O, S, NH, N—R⁵,NH—CO, CO, CH₂CO, S═O and SO₂, or X and Y may form together a groupselected from CH═CH, CO—NR⁵, NH—CO—NH, O—CH(R⁵)—O, NH—CH(R⁵)—O andNH—CH(R⁵)—NH, where the hydrogen in the above groups may optionally besubstituted by an alkyl group;

[0041] R¹, R², R³and R⁴ are each independently selected from H, andsubstituted or unsubstituted alkyl,

[0042] R⁵, R⁶, R⁷ and R⁸ are each independently selected from H,substituted or unsubstituted alkyl, cycloalkyl, aryl, aralsyl,hetroaryl, heterocyclyl, hetroaryl, acyl, carboxyaryl, carboxyalkyl,side chains of naturally and artificially occurring amino acids as wellas derivatives and mimics of such side chains, and linear or cyclicpeptide;

[0043] L is selected from H, OH, NH₂, NHR⁵, a peptide and a solidsupport where R⁵ is as defined above, and

[0044] n and m are integers from 1 to 6, with the exclusion of thefollowing compound: glycinamide,L-tyrosyl-N-[2-[(2S)-4-[(1S)-1-carboxy-3-methylbutyl]-3,4,5,8-tetrahydro-3-oxo-2-(phenymethyl)-1,4-diazocin-1(2H)-yl]2-oxoethyl.

[0045] According to another preferred embodiment, in the above compoundsof formulae I, II, III and IV, X and Y may form together a groupselected from CO—NR⁵, NH—CO—NH, O—CH(R⁵)—O, NH—CH(R⁵)—O andNH—CH(R⁵)—NH, where the hydrogen in the above groups may be substitutedby an alkyl group.

[0046] The following combinations for X and Y are preferred: both X andY are S; or X is NH and Y is CO; or X is CO and Y is NH; or X is NH andY is CH₂; or X is CH₂ and Y is NH; or X and Y are NH—CO—NH or X is N—R⁴and Y is CO; or (vi) X is CO and Y is N—R⁴.

[0047] The invention also provides, according to another of its aspects,a combinatorial library comprising two or more, preferably a plurality,of compounds of any one of the formulae (B), I, II, III or IV. Thelibrary of the invention serves as a readily accessible source ofdiverse macrocyclic compounds for use in identifying new biologicallyactive macrocyclic compounds through pharmaceutical and veterinarycandidate screening assays, for the development of highly effective andenvironmentally friendly insect control and crop control agents, for usein studies defining structure/activity relationships, and/or for use inclinical investigation.

[0048] The selection of an active candidate is preferably achieved froma library of compounds that have the same substituents in the samepositions along the scaffold but the scaffolds differ from each other insize and chirality of the substituents and therefore in theirconformation. The libraries are prepared by the multiple simultaneoussolid phase method [Hougten R. A.,1985] or its automated version, andcontain the calculated number of diversity possibilities. Libraries aretypically synthesized in a 12-48 format, namely each library typicallycontains 12-48 members. Each member of the library will becharacterized, purified and subjected to biological assay. The presentinvention also provides a pharmaceutical composition comprising apharmaceutically acceptable carrier and an effective amount of acompound of the formula I, II, III or IV, as defined above. The newcompounds of the invention may act as modulators of the activity ofcells which activity is mediated by proteins or peptides as will beexplained below.

[0049] The term “effective amount” refers to an amount capable ofdecreasing, to a measurable effect, at least one adverse manifestationof the disease and should be chosen in accordance with the drug used,the mode of administrations the age and weight of the patient, theseverity of the disease, etc.

[0050] Also provided by the invention is use of a compound of theformula I, II, III or IV, as defined above, for the preparation of apharmaceutical composition.

[0051] In addition, the present invention provides a method formodulating a protein or peptide-mediated cell activity, such methodcomprising contacting a compound of the formula I, II, III or IV, asdefined above, with a cell or cellular component having said peptide orprotein. The cell may be from an eukaryotic or prokaryotic organism,from a uni- or multi-cellular organism and may be from plant, bacteriaor animal. The cellular component is selected from cellular organellssuch as nucleous, ribosomes, mitochondria and cell membranes or fromcellular molecules such as receptors, enzymes, substrates, ligands andthe like.

[0052] Examples of peptide or protein-mediated cell activities which aremodulated by the compounds of the invention are: proliferation,differentiation, cellular shape alteration, cellular elongation, uptakeof substances by cells (glucose, neurotransmitters), secretion ofsubstances, cellular metabolism, expression of various proteins.

[0053] Also provided by the present invention, a method for thetreatment of a disease, disorder or condition wherein a therapeuticallybeneficial effect may be evident by the modulation of a protein orpeptide-mediated cell activity, the method comprising: administering toa subject in need of such treatment a therapeutically effective amountof a compound of formula I, II, III or IV.

[0054] In accordance with another embodiment of the invention, thecompound of the invention may be bound to a detectable label such as afluorescence-emitting moiety, a radio-label, a label capable ofundergoing an enzymatic reaction producing a detectable color, a markerfor x-ray, MRI, radio-isotope imaging or PET scan, to produce a labeledadduct. Then, upon administration of such labeled adduct, it may bedetected at a desired location by any manner known in the art and inaccordance with the specific label used, for example, fluorescence,radioactive emission, or a color production, MRI, x-ray and the like.

[0055] The term “bound” refers to covalent or non-covalent (e.g.,electrostatic) binding, which connects the compound of the invention tothe detectable label. Alternatively, the compound of the invention mayhave inherent detectable properties of its own, that enable it to bedetected by any of the above mentioned techniques.

[0056] The present invention is also directed, according to a furtheraspect thereof, to a method for designing new compound libraries thathave novel type of structural complexity and diversity, and can bescreened to identify potent modulators of protein or peptide-mediatedcell activity, so as to develop lead compounds for pharmaceutical,veterinary or agricultural research. Molecules having a molecular weightof up to about 1000 daltons, i.e. small molecules, are preferable.

[0057] More specifically, the present invention provides a method ofidentifying a candidate, which modulates a protein or peptide-mediatedcell activity, the method comprising:

[0058] (a) identifying in said protein or peptide, a domain which isessential for said activity,

[0059] (b) identifying in said domain, pharmacophors essential for theactivity,

[0060] (c) planning a combinatorial library of cyclic compounds havingthe formula I, II, III or IV, as defined above, wherein each suchcompound comprises at least two of the pharmacophors identified in step(b) above or mimics or derivatives of the pharmacophors, where eachmember of the library differs from the other by at least one of thefollowing: i) the size of the ring; ii) the order in which thepharmacophors are arranged in the ring; iii) the chemical nature of thering; iv) the chemical nature of the pharmacophors; v) the chirality ofthe linker between the ring and the pharmacophor; and vi) the chiralityof the pharmacophor,

[0061] (d) synthesizing a plurality of compounds, such compounds beingamong the compounds of the combinatorial library planned in step (c);

[0062] (e) screening the synthesized compounds of step (d) forcandidates that modulate said activity.

[0063] The term pharmacophor refers to the ensemble of steric andelectronic features that is necessary to ensure the optimal molecularinteractions with a specific biological target structure and to trigger(or to block) its biological response. In the present invention the termrefers to those moieties of the side chain or backbone of the peptide orprotein (which mediates the cell activity), that are necessary for thebinding to the other cellular components, the binding eliciting abiological response. The pharmacophor may be a chemical moiety presenton a single side chain or a collection, of chemical moieties present inspatially adjacent side chains.

[0064] The above method may also be used in order to identify a compoundwhich modulates a protein or peptide-mediated cell activity. In suchcase the method comprises the following additional steps after step (e):

[0065] (f) collecting those compounds which modulate said activity in atest assay as compared to the modulation in the same test assay in theabsence of said compound, thereby obtaining modulators of a protein orpeptide-mediated cell activity; and

[0066] (g) producing the compounds selected in step (f) above.

[0067] The present invention further provides a compound which modulatesa protein or peptide-mediated cell activity obtained by the abovemethod.

[0068] The present invention may also be utilized in agriculture.Therefore, the present invention also provides a method for thediscovery of new agents for use in agriculture, wherein such agents arebased on modulators of proteins or peptides derived from insects orplants.

GLOSSARY

[0069] Definitions

[0070] A “library” is a collection of compounds which while sharing somecommon structural elements (which may be common scaffolds, common ringsizes, common substituents and the like), are diverse from each other byat least one of the following properties: i) the size of the ring; ii)the order in which the pharmacophors are arranged in the ring; iii) thechemical nature of the ring; iv) the chemical nature of thepharmacophors; v) the chirality of the linker between the ring and thepharmacophor; and vi) the chirality of the pharmacophor. The libraryallows screening from among a plurality of compounds for those that havea desired property. The library may be designed by a combinatorial orclassical chemical process.

[0071] A “lead compound” is a library compound in a selectedcombinatorial library, for which the assay has revealed significanteffect relevant to a desired cell activity to be modulated. In thepresent case the property is the modulator of at least one peptide orprotein-mediated activity.

[0072] “Peptide or protein-mediated cell activity” refers to aphysiological property of a cell that is caused, directly or indirectly(the latter referring to an effect caused by an, effector which is moredownstream in the pathway) by the interaction between a protein orpeptide and another cellular component (the term “cellular component”including: other proteins or peptides of the same or different types,membranes, nucleic acids, lipoproteins, nucleotides, co-factors,hormones, ion effectors and the like). The interaction between theprotein and the other cellular component may be of the type:receptor-ligand, enzyme-substrate, DNA-binding proteins-DNA etc. Saidinteraction mediates (causes) directly or indirectly a cell activitysuch as: expression of a protein, proliferation, differentiation,cell-elongation, cell-shape alteration, cellular metabolism, cellularupdate of external substances, secretion of substances from the cellsand the like.

[0073] “Modulate/Modulator” refers to increase or decrease in at leastone peptide or protein-mediated cell activity, in the presence of thecompound of the invention, or to the change of the response of the cellto the presence of a physiological cue, as compared to the activity orresponse, respectively, in the absence of the compound. Examples of suchphysiological cues are presence of effectors, the modulation being achange in the cellular response to a ligand, hormone, response to toxicsubstances (pesticides), stress (heat shock, draught, lack of nutrients)aging and the like.

[0074] The term “substituents” refers to chemical radicals which arebonded to or incorporated onto the scaffold through the synthesisprocess of the library. The different functional groups account for thediversity of the molecules throughout the library and are selected toimpart diversity of structure, function and biological activity to thescaffold in the case of diverse libraries, and optimization of aparticular biological activity in the case of directed libraries.

[0075] “Aryl” means one or more aromatic rings, each of 5 or 6 ringcarbon atoms and includes substituted aryl having one or morenon-interfering substituents. Multiple aryl rings may be fused, as innaphthyl, or unfused, as in biphenyl.

[0076] “Alkyl” means straight or branched chain or cyclic hydrocarbonhaving 1 to 10 carbon atoms.

[0077] “Substituted alkyl”, is alkyl having one or more non-interferingsubstituents.

[0078] “Halo” means chloro, fluoro, iodo or bromo.

[0079] “Heterocycle” or “heterocyclic” means one or more rings of 5, 6or 7 atoms with or without unsaturation or aromatic character,optionally substituted with one or more non-interfering substituents,and at least one ring atom which is not carbon. Preferred heteroatomsinclude sulfur, oxygen, and nitrogen. Multiple rings may be fused, as inquinoline or benzofuran, or unfused as in 4-phenylpyridine. Suitablesubstituents on the heterocyclic ring structure include, but are notlimited to halo, C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, C1-C10alkoxy, C7-C12 aralkyl, C7-C12 alkaryl, C1-C10 alkylthio, arylthio,aryloxy, arylamino, C3-C10 cycloalkyl, C3-C10 cycloalkenyl,di(C1-C10)-alkylamino, C2-C12 alkoxyalkyl, C1-C6 alkylsulfinyl, C1-C10alkylsulfonyl, arylsulfonyl,aryl, hydroxy, hydroxy(C1-C10)alkyl,aryloxy(C1-C10)alkyl, C1-C10 alkoxycarbonyl, aryloxycarbonyl,aryloyloxy, substituted alkoxy, fluoroalkyl, nitro, cyano,cyano(C1-C10)alkyl, C1-C10 alkanamido, aryloylamido, arylaminosulfonyl,sulfonamido, amidino, carbamido, carboxy, heterocyclic radical,nitroalkyl, and —(CH₂)_(m)—Z—(C1-C10 alkyl), where m is 1 to 8 and z isoxygen or sulfur.

[0080] The term “solid support” refers to a solvent insoluble materialhaving cleavable covalent bonds for use in preparing the librarycompounds of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0081] Many biological processes are critically dependent onprotein:protein, protein:peptide and protein:nucleic acid interactions,and many drugs are small molecules known to disrupt such interactions(antagonists) or alternatively mimic one component of the interaction insuch a manner so that the activity controlled by the interaction cantake place in the presence of the drug and the other cellular component(agonist).

[0082] The drugs which work by interruption of such interactions (forexample by the interruption of a receptor-ligand interaction) mimic infact a domain of one of the proteins participating in the interaction.By one option this mimic creates an antagonist that competes with theprotein for binding with the other member of the interaction (the othercellular component), leading to decrease in the interaction and hencedecrease of the cell activity controlled or caused (directly orindirectly) by the interaction. Where the cell activity is an “on”physiological process, (for example, increase in production of anagent), the interruption will close the “on” reaction and decrease thephysiological process (decrease production of the agent). Where the cellactivity is an “off” reaction (for example a signal causing inhibitionof proliferation) th interruption will close the “off” reaction and willincrease the physiological process, for example, cause increasedproliferation.

[0083] By another option the drug may work as an agonist and cause themodulation of the cell activity by mimicking, the protein (that isessential for the cell activity) in the interaction in such a mannerthat the cell activity takes place as if the native protein and not thecompound were interaction with the other cellular component. For examplethe compound may be able to activate the cellular component with whichthe protein interacts in a similar way to the protein itself.

[0084] The aim of the compounds of the present invention is to mimic aregion in one of the participators of the interaction, so as to competefor the binding on the other participator of the interaction (either inthe antagonist or the agonist manner) thus changing the interaction andleading to a change in the physiological process or property controlledby the interaction.

[0085] The rational for the present invention is the following: manylibraries used for the discovery of drug leads are composed ofheterocyclic scaffolds that are too constrained (rigid) to allowconformational complementarity essential for the interactions withproteins, peptides, polysaccharides or nucleic acids. The combinatoriallibrary of the invention allows the generation of sub-libraries withspatial diversity, which is obtained by the diversity in ring size andchirality of the link between the substituent and the scaffold. Thisresults in a library where each individual scaffold has a differentflexibility ad a different spatial positioning of the pharmacophor. Thedesign of the library of the invention increases the probability thatsome members of the library have the ability to undergo conformationalcomplementarily, i.e. the pharmacophors are present in the correctorientation to interrupt or mimic the interaction with the othercellular component. The present invention allows the design andsynthesis of libraries which occupy a larger proportion of the“probability space” of the pharmacophor positioning (i.e. increase theprobability of the substituents to be present in varying positions inthe space, thus increasing the probability that at least onepositioning-conformation is the bioactive conformation,) while stillcreating relatively small, focused libraries. These properties lead tofast discovery and optimization of novel drug leads.

[0086] The classical elements of diversity of state-of-the-art,currently available macrocyclic, i.e. scaffold based libraries are basedmainly on:

[0087] (i)The chemical nature of the scaffold;

[0088] (2)The size and chemical nature of the linkers that connectbetween the scaffold and the various substituents;

[0089] (3)The chemical nature of the substituents;

[0090] (4) The order in which the substituents are arranged on thescaffold.

[0091] The libraries of the new compounds of the present inventioncomprise a novel element of diversity, namely spatial diversity, thatresults from the varying size of the scaffold ring, the chirality of thelinker or from a combination of the two. This diversity is new in thefield of small molecule combinatorial chemistry.

[0092] Spatial diversity is defined as diversity elements that alter theconformation of the compounds, which in fact lead to altered spatialpositioning of the phamacophors. The present invention deals withlibraries of compounds having new elements of diversity, namely spatialdiversity elements: the chirality of the linkers (which effects thespatial arrangement of the substituents) and the size of the ring. Themembers of the library of the invention may also differ from each otherin the classical elements of diversity (1)-(4) mentioned above.

[0093] According to a specific embodiment of the present invention, allthe members in a library have the same elements of diversity (1)-(4)mentioned above: the same scaffold with the same composition and orderof atoms within. the scaffold; the same linker with the same size andchemical nature; the same substituents/pharmacophors arranged in thesame order on the scaffold, but they differ from each other in the sizeof the scaffold and/or the chirality of the linkers. This in turndetermines the possible conformational (spatial) positioning of thepharmacophor of each compound and allows for the selection of the leadcompound having the appropriate ability of conformationalcomplementarity. The libraries of the invention are composed of a seriesof compounds that differ from each other by an incremental alteration oftheir possible conformations. Thus, every library covers an entire rangeof the conformational probabilities and increases the chances ofobtaining a compound with at least one bioactive conformation.

[0094] Design of the libraries

[0095] The method of the present invention, for identifying a candidatewhich modulates a protein or peptide-mediated cell activity, comprisesthe following steps:

[0096] (a) identifying in said protein or peptide, a domain essentialfor said activity,

[0097] (b) identifying in said domain, pharmacophors essential for theactivity,

[0098] c) planning a combinatorial library of cyclic compounds havingthe formula I, II, III or IV as defined above, wherein each suchcompound comprises at least two of the pharmacophors identified in step(b) above or mimics or derivatives of the pharmacophors, where eachmember of the library differs from the other by at least one of thefollowing: i) the size of the ring; ii) the order in which thepharmacophors are arranged in the ring; iii) the chemical nature of thering; iv) the chemical nature of the pharmacophors; v) the chirality ofthe linker between the ring and the pharmacophor; and vi) the chiralityof the pharmacophor;

[0099] (d) synthesizing a plurality of compounds from the combinatoriallibrary planned in step (c);

[0100] (e) screening the compounds synthesized in step (d) forcandidates that modulate said activity.

[0101] Between the step (c) of planning of the fill library and step (d)of synthesizing a plurality of compounds from the library (which mayform together the full library or a part of the library), it is possibleto add a step of virtually screening the library to identify thosecompounds which are better mimics of the domain than others. Suchvirtual screening can help and predict which compounds have a betterchance of assuming the bioactive conformation and it is preferable tostart the screening with the compounds that are, according to 3Dmodeling the most likely mimics.

[0102] The above steps are showed schematically in the following chart:

[0103] As the compounds of the invention intend to mimic a domain in aprotein, so as to interrupt or to mimic its interaction with othercellular components and thus modulate the cell activity (mediated by theprotein), it is desired that they resemble the desired domain and thepositioning of the pharmacophors in the domain of the protein (whichpharmacophors they mimic) as close as possible. Therefore, when decidingat the library planning step how to produce the best library, and at thesynthesizing step, which of the members of the planned library should besynthesized, the following questions should be asked:

[0104] 1. Are the relevant pharmacophors (or derivatives or mimics ofthe pharmacophors) present in the planned library?

[0105] 2. Is the order of the pharmacophors on the scaffold and thedistance of the pharmacophores from each other, suitable for achieving asuitable bioactive conformation (correct positioning of the

[0106] 3. Is the compound capable in one of its conformation ofattaining the correct positioning of the pharmacophors?

[0107] 4. Is the possible conformation energetically favorable?

[0108] 5. Is there a certain degree of conformation flexibility to allowconformational complementarity?

[0109] Most of the above questions can be answered during the planningstage and the synthesis decision stage on a computer using commerciallyavailable bioinformatic programs such as Tripose™.

[0110] The coordinates of amino acid side chains of a protein can beobtained from the Protein Data Bank (PDB) files. This data is based onthe 3D structure of the protein (preferably as a complex with theappropriate ligand) either obtained by crystallography or homologymodeling. The 3D information allows to identify the exposed side chainsand these accesible side chains are possible pharmacophors. In cases ofproteins for which the 3D structure was not determined, the essentialamino acids within a protein may be determined by the method ofcombinatorial alanine-scanning (Morrison and Weiss, (2001) Curr. Opin.Chem. Biol. 5, 302-307), also known as Ala-Scan. Another method is knownas omission libraries and is described in Campian et al. (1998) Bioorg.Med. Chem. Lett. 8, 2357. Yet other methods are site directedmutagenesis and protein engineering (Winter et al (1982)). The aminoacids and backbone elements essential for a certain function may bedivided into two categories: those who interact with the receptiveprotein, nucleic acid, polysaccharide or cell membrane and thoseresponsible for the conformation of the essential region. The sidechains and backbone elements of the former are those that participate inthe creation of pharmacophors and the present invention relates to thecreation of such pharmacophors, or their mimics and their incorporationin the scaffolds of the invention for the purpose of creating a mimic ofa region of the protein.

[0111] As mentioned above, the essential amino acids within a proteinmay be determined by the method of combinatorial alanine-scanning.Alanine scanning, a method of systematic and sequential alaninesubstitution, has been particularly useful for the identification ofpharmacophors in a given peptide sequence. This method is based on thesynthesis of a library in which each amino acid residue in a peptidechain is sequentially replaced by alanine, and biological screening ofthe library. Substitution of functional amino acid residues by themethyl group of alanine leads to the removal of all the side chain atomspast the β-carbon. Thus, the role of side-chain functional groups atspecific position can be inferred. Alanine residue have the samebackbone dihedral angles as other functional residues and thus thebackbone conformation is not drastically perturbed by such substitution,as would be the case in glycine scan libraries. In this case, the sidechain is nullified, which leads to the introduction of flexibility intothe peptide backbone.

[0112] An additional method for the elucidation of pharmacophors is thesynthesis and biological screening of omission libraries. Omissionlibraries, based on a given peptide sequence is a library that containall the possible peptides that compose the parent peptide. Omissionlibrary is divided into sequential and non sequential. In, sequentialomission library, amino acids are omitted from the carboxy- and amino-ends, whereas in non sequential library amino acids are omitted from theinterior of the peptide sequence. Thus, sequential omission librarybased on a hexapeptide contains 2 pentapeptides, 3 tetrapeptides, 4tripeptides and 5 dipeptides (total of 14 peptides). Non-sequentialomission library based on a hexapeptide contains 4 pentapeptides, 18tetrapeptides, 27 tripeptides and 9 dipeptides (total of 58 peptides).Beside information on essential pharmacophors, omission libraries canfinish shorter active peptides that will facilitate the design, oflibraries.

[0113] Once the pharmacophors are determined they are incorporated intothe new compounds of the invention as one of the R substituents, toproduce combinatorial libraries that will permit, alter undergoingscreening with suitable assays to screen for and to select candidatesfor a variety of uses in human medicine, veterinary medicine and inagriculture. Each compound may bind at least two pharmacophors, usuallyat least three pharmacophors and typically up to six, preferably up tofive pharmacophors. Preferably the compound contain three to five suchpharmacophors, attached to the scaffold either directly or through alinker. At times, when more then four pharmacophors have to be linked toa molecule, this can be achieved through binding the additional fifth,sixth etc. pharmacophors to the pharmacophors that are already connectedto the scaffold.

[0114] In conclusion, the method of the invention utilizes spatiallibraries that can generate novel leads for the disruption ofprotein:protein, protein:peptide, protein:cell membrane andprotein:nucleic acid interactions, in animals and plants.

[0115] The pharmaceutical composition of the invention may beadministered by any of the known administration routes, inter alga,oral, intravenous, intraperitoneal, intramuscular, subcutaneous,sublingual, intraocular, intranasal or topical administration routes.Appropriate unit dosage forms of a stration include the forms for oraladministration, such as tablets, capsules, powders, granulates and oralsolutions or suspensions and the forms for sublingual and buccaladministration, the forms for parenteral administration useful for asubcutaneous, intramuscular or intravenous injection, as well as theforms for rectal administration.

[0116] The carrier should be selected in accordance with the desiredmode of administration and include any known components, e.g. solvents;emulgators, excipients, talc; flavors; colors, etc. The pharmaceuticalcomposition may comprise, if desired, also other pharmaceutically-activecompounds which are used to treat the disease, eliminate side effects oraugment the activity of the active component.

[0117] In the case of tablets for oral use, carriers which are commonlyused include lactose and corn starch. Lubricating agents, such asmagnesium stearate, are also typically added. For oral administration ina capsule form, useful diluents include lactose and dried corn starch.When aqueous suspensions are administered orally, the active ingredientis combined with emulsifying and suspending agents. If desired, certainsweetening and/or flavoring and/or coloring agents may be added.

[0118] Typically, the pharmaceutical compositions of this invention willbe administered from about 1 to about 5 times per day or alternatively,as a continuous infusion. A typical preparation will contain from about5% to about 95% active compound (w1w). Preferably, such preparationscontain from about 20% to about 80% active compound. As the skilledartisan will appreciate, lower or higher doses than those recited abovemay be required. Specific dosage and treatment regimens for anyparticular patient will depend upon a variety of factors, including theactivity of the specific compound employed, the age, body weight,general health status, sex, diet, time of administration, rate ofexcretion, drug combination, the patients disposition to the diseasestate and the judgment of the treating physician. In general, thecompound is most desirably administered at a concentration level thatwill generally afford effective results without causing any harmful ordeleterious side effects.

[0119] The pharmaceutical composition may comprise, if desired, alsoother pharmaceutically-active compounds which are used to treat thedisease, eliminate side effects or augment the activity of the activecomponent.

[0120] Synthetic Approach

[0121] In general, the compounds of the invention are prepared accordingto the routes showed in Schemes 1-4 below. The solid phase synthesis ofscaffolds I-IV comprise of a series of couplings of the appropriateprotected acids and reductive alkylations with ω-functionalizedprotected aldehydes. The assembly of the appropriate linear scaffold onthe solid support is followed by removal of the protecting groups P₁ andP₂ and cyclization. The appropriate scaffold is obtained afterdeprotection-removal from the solid support.

EXAMPLES

[0122] Synthesis

[0123] A library composed of 26 molecules that have the formula I andwherein R¹ and R² are either benzyl (side chain of phenylalanine) orhydroxybenzyl (side chain of tyrosine) and R³ is benzyloxycarbonyl(which is a mimic of the side chain of phenylalanine) was synthesizedand characterized. The library was synthesized by the SimultaneousMultiple Solid Phase methodology (Houghten (1985) Proc. Natl. Acad. Sci.USA, 82 5131) as showed in Scheme 5 below. The molecules werecharacterized by HPLC, MS and MS-MS spectrometry.

[0124] Synthetic Procedures According to Scheme 5 Above:

[0125] Rink amide MBHA resin [Rink H. (1987)] (0.1 g in each bag, 0.6mmol/g) was preswollen for 2 h in NMP while shaking in reaction vesselequipped with sintered glass bottom. The Fmoc protecting group wasremoved from the resin by reaction with 20% piperidine in NMP (2×30min). Fmoc removal was monitored by chloranil test. A coupling cycle wascarried out with Fmoc-AA (AA is abbreviation of amino acid) (5 eq), BTC(1.65 eq), and 2,4,6 colidine (14 eq) in DCM for 2 h at roomtemperature. Reaction completion was monitored by qualitative chloraniltest. Following coupling the peptidyl-resin was washed with DCM (×5) andfor 2 mm. Fmoc removal and washing steps were carried out as describedabove. Fmoc removal was monitored by the chloranil test.

[0126] The peptidyl-resin was then washed by a mixture of NMP: MeOH1:1/1% and a solution of the aldehyde (1 eq) in the mixture above wasadded (10 ml for 12 bags). Then additional 40 ml of this mixture wasadded and the mixture was shaken for 5 min. Taken, 2 eq of NaBH₃CN wereadded and the reaction vessel was shaken for 2 h. The resin was washedas follows: DCM (2×2 min), EtOH (2×2 min), NW (2×2 min), DCM (3×2 min).(Chloranil test gave blue color immediately). The following coupling wasperformed using Fmoc-AA (5 eq), BTC (1.65 eq) and 2, 4, 6 colidine (14eq) in dibromomethane at 50÷C. for 2 h and was repeated when necessary.Fmoc detection and washing steps were carried out as described above.Reductive alkylation and washing steps were carried out as describedabove. A solution of benzylcholorformate (6 eq), and DIEA (12 eq) in DMFwas added to the resin and the mixture was shaken for 1 h. The reactionwas repeated and ten the resin was washed with NMP (5×2 min) and DCM(2×2 min). Reaction completion was monitored by chloranil test.

[0127] Disulfide Bridge Formation

[0128] The disulfide bridge was oxidized using iodine (10 eq) in DCM andshaking for 3 h. The resin was washed as follows: DMF (2×2 min), 2%ascorbic acid in DMF (2×2 min), NMP (5×2 min), DCM (4×2 min).

[0129] Analytical Procedures

[0130] All the crude compounds were analyzed by MS and analyticalreversed-phase HPLC (RP18 Vydak 4×250 mm; flow: 1 mL/min.; T=30° C.;detection UV 214 nm; gradient: A=0.1% TFA in TDW, B=0.1% TFA in CH₃CN, 0min 95:5, 5 min 95:5, 33 min 5:95, 38 min 95:5, 42 min 95:5).

[0131] The molecules were purified by preparative reversed-phase HPLC(RP18 Vydak. 2.5×250 mm; flow: 9 mL/min.; T=30° C.; detection UV 214 nm;gradient: A=0.1% TFA in TDW, B=0.1% TFA in CH₃CN, 0 min 95:5, 5 min95:5, 33 min 5:95, 38 min 95:5, 42 min. 95:5).

[0132] Fractions were collected, lyophilized and characterized byanalytical HPLC and MS analysis. Results are shown. in Table 1 below.

[0133] Synthesis of Aldeydes

[0134] Trityl-thiopropanal

[0135] a) Trityl-thiopropanoic Acid

[0136] Trityl mercaptan (36.13 g, 0.131 mol) was added stepwise to asuspension of NaH (11.5 g, 60% in mineral oil 0.288 mol) in 80 mL DMFunder cooling and nitrogen atmosphere, the reaction mixture was stirredminutes after the is addition was completed. Then, a solution ofbromopropionic acid (20 g, 0.131 mol) dissolved in 50 mL DMF was addedstepwise. After the addition was completed the reaction mixture wasstirred for 30 minutes and then cooling and nitrogen atmosphere werestopped and the reaction mixture was sealed and left overnight. Then,500 mL chloroform were added and the mixture was washed with 4×200 mL ofsaturated solution of KHSO₄ and 4×200 mL TDW (the solid that precipitateduring the washings should also be collected with the organic layer).The organic layer was evaporated and the product (that contained DMFtraces) was precipitated by adding 300 mL DW and stirring for fewminutes. The product was collected by filtration and dried by suctionand then in vaccuo. The crude product was purified as follows: 150 mL ofCHCl₃ were added to the white solid and the mixture was stirred for fewminutes. Then 200 mL of PE 40-60 were added and the solid was collectedby filtration yielding 37.61 g (82% yield) of white powder, mp 177-183°C., ¹H NMR (CDCl₃, 300 MHz, 298K) δ2.24 (t, 2H), 2.46 (t, 2H), 7.18-7.48(m, 15H). MS (ES) m/z 347.

[0137] b) Trityl-thiopropanoic Acid Hydroxamate

[0138] A solution of N,O dimethylhydroxylamine hydrochloride (2.188 g,0.0225 mol) in 40 mL DMF was added to a mixture of 6.96 g (0.02 mol) ofTrityl-thiopropanoic acid and PyBoP (11.45 g, 0.022 mol). DIEA (10.4 mL,0.06 mol) was added and the clear solution was stirred for 3 hours. EA(120 mL) was added to the stirred solution followed by 240 mL ofsaturated bicarbonate solution. The organic layer was collected andwashed with additional two portions of 100 mL of saturated bicarbonatesolution, 100 mL of TDW, 2×100 mL KHSO₄ 1M, and 100 mL TDW, dried overNa₂SO₄ and evaporated to dryness yielding 10.36 g (92% yield) of yellowoil. ¹H NMR (CDCl₃, 300 MHz) δ2.38 (t, 2H), 2.51 (t, 2H), 3.10 (s, 3H),3.56 (s, 3H), 7.15-7.50 (m, 15H).

[0139] c) Trityl-thiopropanal

[0140] LiAlH₄ (2.014 g, 0.053 mol) was added in portions to a solutionof 10.36 g (0.0265 mol) of Trityl-thiopropanoic acid hydroxamate in 260mL dry diethyl ether under cooling in ice bath and argon atmosphere. Thereaction mixture was stirred for 2 hours (monitored by TLC PE:EA=1:1).560 mL of EA were added followed by addition of 560 mL of KHSO₄ 1M. Themixture was stirred for additional 30 minutes. The organic layer wascollected and washed with 390 mL of KHSO₄ 1M and 390 mL of saturatedNaCl, dried over Na₂SO₄ and evaporated yielding 7.68 g (87% yield) ofwhite solid. ¹H NMR (CDCl₃, 300 MHz, 298K) δ2.36 (t, 2H), 2.46 (t, 2H),7.15-7.50 (m, 15H), 9.55 (t, 1H).

[0141] Trityl thiobutyral

[0142] a) Trityl-thiobutyric Acid;

[0143] Trityl mercaptar (36.13 g, 0.131 mol) was added stepwise to asuspension of NaH (11.5 g, 60% in mineral oil 0.288 mol) in 100 mL DWMunder cooling and nitrogen atmosphere, the reaction mixture was stirredminutes after the addition was completed. Then, a solution ofbromobutyric acid (21.88 g, 0.131 mol) dissolved in 150 mL DMF was addedstepwise. After the addition was completed the reaction mixture wasstirred for minutes and then cooling and nitrogen atmosphere werestopped and the reaction mixture was sealed and left overnight. Then,500 mL chloroform were added and the mixture was washed with 4×200 mL ofsaturated solution of KHSO₄ and 4×300 mL of water (the solid thatprecipitate during the washings should also be collected with theorganic layer). The organic layer was evaporated and the oily product(that contained DMF traces) was triturated by adding 300 mL TDW andsting vigorously for few minutes. The product was collected byfiltration, washed by TDW and dried by suction. The crude product waspurified as follows: 200 mL of PE was added to the white solid and themixture was stirred for 15 minutes. The solid was collected byfiltration and dried in vaccuo yielding 36.82 g (77.6% yield) of whitepowder. ¹H NMR (CDCl₃, 300 MHz, 298K) δ1.67 (m, 2H), 2.22 (t, 2H), 2.30(t, 2H), 7.10-7.50 (m, 15H).

[0144] b) Trityl-thiobutyric Acid Hydroxamate

[0145] A solution of N,O dimethylhydroxylamine hydrochloride (0.83 g0.0084 mol) in 20 mL DMF was added to a mixture of 2.77 g (0.0076 mol)of Trityl-thiobutyric acid and PyBoP (4.39 g, 0.0084 mol). DIEA (4 mL,0.023 mol) was added and the clear solution was stirred for 3 hours (pHshould be monitored and kept basic). EA (50 mL) was added to the stirredsolution followed by 90 mL of saturated bicarbonate solution. Theorganic layer was collected and washed with additional two portions of40 mL of sated bicarbonate solution, 40 mL of water, 2×40 mL KHSO₄1M,and 40 mL water, dried over Na₂SO₄ and evaporated to dryness yielding3.06 g (quantitative yield) of yellow oil. ¹H NMR (CDCl₃, 300 MHz) δ1.74(s, 2H), 2.23 (t, 2H), 2.38 (t, 2H), 3.13 (s, 3H), 3.63 (s, 3H),7.10-7.50 (m, 15H).

[0146] c) Trityl-thiobutanal

[0147] LiAlH₄ (0.574 g, 0.0151 mol) was added in portions to a solutionof 3.06 g (0.0075 mol) of the hydroxamate in 100 mL dry diethyl etherunder cooling in ice bath and argon atmosphere. The reaction mixture wasstirred for 1 hour (monitored by TLC PE:EA=1:1). 150 mL of EA were addedfollowed by addition of 150 mL of KHSO₄ 1M. The mixture was stirred foradditional 30 minutes. The organic layer was collected and washed with100 mL of KHSO₄ 1M and 100 mL of saturated NaCl, dried over Na₂SO₄ andevaporated yielding 1.90 g (73% yield) of white solid. ¹H NMR (CDCl₃,300 MHz, 298K) δ1.66 (m, 2H), 2.22 (t, 2H), 2.38 (t, 2H) 7.15-7.50 (m,15H), 9.61 (1H).

[0148] Trityl Thiovaleric Aldehyde

[0149] a) Trityl-thiovaleric Acid

[0150] Trityl mercapta (36.13 g, 0.131 mol) was added stepwise to asuspension of NaH (11.5 g, 60% in mineral oil 0.288 mol) in 100 mL DMFunder cooling and nitrogen atmosphere, the reaction mixture was stirredminutes after the addition was completed. Then, a solution ofbromovaleric acid (23.71 g, 0.131 mol) dissolved in 150 mL DW was addedstepwise. After the addition was completed the reaction mixture wasstirred for 30 minutes and then cooling and nitrogen atmosphere werestopped and the reaction mixture was sealed and left overnight. Then,500 mL chloroform were added and the mixture was washed with 4×200 mL ofsaturated solution of KHSO₄ and 4×300 mL of water (the solid thatprecipitate during the washings should also be collected with theorganic layer). The organic layer was evaporated resulting in a solidproduct (that contained DMF traces). 300 mL TDW were added and themixture was stirred vigorously for few minutes. The product wascollected by filtration and partially dried by suction. The crudeproduct was purified as follows: 200 mL of PE was added to the whitesolid and the mixture was stirred for a few minutes. The solid wascollected by filtration and dried in vaccuo yielding 45.06 g (91% yield)of white powder. ¹H NMR (CDCl₃, 300 MHz, 298K) δ1.41 (m, 2H), 1.58 (m,2H), 2.18 (m., 4H), 7.15-7.45 (m, 15H). MS (ES) m/z 376.

[0151] b) Trityl-thiovaleric Acid Hydroxamate

[0152] A solution of N,O dimethylhydroxylamine hydrochloride (0.7 g,0.0071 mol) in 16 mL DW was added to a mixture of 2.45 g (0.0065 mol) ofTrityl-thiovaleric acid and PyBoP (3.73 g, 0.0071 mol). DIEA (3.4 mL,0.02 mol) was added and the clear solution was stirred for 3 hours (pHshould be monitored and kept basic). EA (50 mL) was added to the stirredsolution followed by 90 mL of saturated bicarbonate solution. Theorganic layer was collected and washed with additional two portions of40 mL of saturated bicarbonate solution, 40 mL of water, 2×40 mLKHSO₄1M, and 40 mL water, dried over Na₂SO₄ and evaporated to drynessyielding 3.06 g (quantitive yield) of yellow oil. ¹H NMR (CDCl₃, 300MHz) δ1.74 (m 2H, 2.23 (t, 2H), 2.38 (t, 2H), 3.13 (s, 3H), 3.63 (s,3H), 7.10-7,50 (m, 15H).

[0153] c) Trityl Thiovaleric Aldehyde

[0154] LiAlH₄ (0.574 g, 0.0151 mol) was added in portions to a solutionof 3.06 g (0.0075 mol) of the hydroxamate in 100 mL dry diethyl etherunder cooling in ice bath and argon atmosphere. The reaction mixture wasstirred for 1 hour (monitored by TLC PE:EA=1:1). 150 mL of EA were addedfollowed by addition of 150 mL of KHSO₄1M. The mixture was stirred foradditional minutes. The organic layer was collected and washed with 100mL of KHSO₄ 1M and 100 mL of saturated NaCl, dried over Na₂SO₄ andevaporated yielding 1.90 g (73% yield) of white solid. ¹H NMR (CDCl₃,300 MHz, 298K) 67 1.66 (m, 2H), 2.22 (t, 2H), 2.38 (t, 2H) 7.15-7.50 (m,15H), 9.61 (t, 1H).

[0155] Trityl Thiohexanal

[0156] a) Trityl-thiohexanoic Acid

[0157] Trityl mercaptan (36.13 g, 0.131 mol) was added stepwise to asuspension of NaH (11.5 g, 60% in mineral oil 0.288 mol) in 100 mL DMFunder cooling and nitrogen atmosphere, the reaction mixture was stirredminutes after the addition was completed. Then, a solution ofbromohexanoic acid (g, 0.128 mol) dissolved in 150 mL, DMF was addedstepwise. After the addition was completed the reaction mixture wasstirred for 30 minutes and then cooling and nitrogen atmosphere werestopped and the reaction mixture was sealed and left overnight. Then,500 mL chloroform were added and the mixture was washed with 4×200 mL ofsaturated solution of KHSO₄ and 4×300 mL of water (the solid thatprecipitate during the washings should also be collected with theorganic layer). The organic layer was evaporated and the product (thatcontained DMF traces) was triturated by adding 300 mL TDW and stirringvigursly for few minutes. The product was collected by filtration,washed by TDW and dried by suction. The crude product was purified asfollows: The solid was dissolved in a mixture of 150 ml of CHCl₃ and 200ml of PE, and the solution was evaporated. The oil obtained wastriturated by addition of 100 ml PE and 50 ml of Et₂O and 50 ml Et2O and50 ml PE. The solid was collected by filtration and dried in vaccuoyielding 33.92 g (68% yield) of white powder. ¹h NMR (CDCl₃, 300 MHz,298K) 6 1.31 (m, 2H), 1.37 (m, 2H), 1.50 (m, 2H), 2.15 (t, 2H), 2.26 (t;2H), 7.15-7.50 (m, 15H).

[0158] b) Trityl-thiohexanoic Acid Hydroxamate

[0159] A solution of N,O dimethylhydroxylamine hydrochloride (1.23 g,0.0126 mol) in 25 mL DMF was added to a mixture of 4.47 g (0.0115 mol)of Trityl-thiohexanoic acid and PyBoP (6.56 g, 0.0126 mol). DIEA (6 mL,0.0344 mol) was added and the clear solution was stirred for 3 hours (pHshould be monitored and kept basic). EA (70 mL) was added to the siredsolution followed by 130 mL of saturated bicarbonate solution. Theorganic layer was collected and washed with additional two portions of60 mL of saturated bicarbonate solution, 60 mL of water, 2×60 mL KHSO₄1M, and 60 mL water, dried over Na₂SO₄ and evaporated to drynessyielding 4.29 g (86% yield) of yellow oil. ¹H NMR (CDCl₃, 300 MHz) 671.29 (m, 2H), 1.44 (m, 2H), 1.51 (m 2H), 2.16 (t, 2H), 2.33 (t, 2H),3.15 (s, 3H), 3.65 (s, 3H) 7.15-7.50 (m, 15H).

[0160] c) Trityl-thiohexanal

[0161] LiAlH4 (0.75 g, 0.0198 mol) was added in portions to a solutionof 4.29 g (0.0099 mol) of the hydroxamate in 130 mL dry diethyl etherunder cooling in ice bath and argon atmosphere. The reaction mixture wasstirred for 1 hour (monitored by TLC PE:EA=1:1). 200 mL of EA were addedfollowed by addition of 200 mL of KHSO₄1M. The mixture was stirred foradditional minutes. The organic layer was collected and washed with 140mL of KHSO₄ 1M and 140 mL of saturated NaCl, dried over Na₂SO₄ andevaporated yielding 3.45 g (93% yield) of white solid. ¹H NMR (CDCl₃,300 MHz, 298K) 67 1.38 (m, 2H), 1.49 (m, 2H), 1.60 (m, 2H), 2.15 (t,2M,) 2.34 (t, 2H), 7.10-7.55 (m, 15I1), 9.71 (t, 1H).

[0162] Trityl thioacetaldehyde and trityl thiopentanal were prepared byprocedures similar to those described above. TABLE 1 structure and MScharacterization of a library according to the present invention, thepreparation of which is showed in Scheme 5 above. compound M.W M.Wnumber R⁶ R⁷ R⁸ m n calc. Obsvd. 1 L-hydroxybenzyl L-Benzyl Z# 4 5649.87 653.3 2 L-hydroxybenzyl L-Benzyl Z 5 4 649.87 653.3 3 L-BenzylD-hydroxybenzyl Z 4 5 649.87 653.2 4 L-Benzyl D-hydroxybenzyl Z 5 4649.87 653.3 5 D-hydroxybenzyl L-Benzyl Z 4 5 649.87 653.2 6D-hydroxybenzyl L-Benzyl Z 5 4 649.87 653.2 7 D-Benzyl L-hydroxybenzyl Z4 5 649.87 653.3 8 D-Benzyl L-hydroxybenzyl Z 5 4 649.87 653.2 9L-hydroxybenzyl D-Benzyl Z 4 5 649.87 653.2* 10 L-hydroxybenzyl D-BenzylZ 5 4 649.87 653.3 11 L-hydroxybenzyl L-hydroxybenzyl Z 6 6 707.94711.79 12 D-hydroxybenzyl D-hydroxybenzyl Z 6 6 707.94 711.85 13L-hydroxybenzyl D-hydroxybenzyl Z 6 6 707.94 711.91 14 D-hydroxybenzylL-hydroxybenzyl Z 6 6 707.94 711.43 15 L-hydroxybenzyl L-Benzyl Z 6 6691.94 694.8 16 D-hydroxybenzyl D-Benzyl Z 6 6 691.94 693.41 17L-hydroxybenzyl D-Benzyl Z 6 6 691.94 693.79 18 D-hydroxybenzyl L-BenzylZ 6 6 691.94 693.91 19 L-Benzyl L-hydroxybenzyl Z 6 6 691.94 693.79 20D-Benzyl D-hydroxybenzyl Z 6 6 691.94 693.6 21 L-Benzyl D-hydroxybenzylZ 6 6 691.94 693.23** 22 D-Benzyl L-hydroxybenzyl Z 6 6 691.94 693.23**23 L-Benzyl L-Benzyl Z 6 6 675.95 677.24** 24 D-Benzyl D-Benzyl Z 6 6675.95 677.23** 25 L-Benzyl D-Benzyl Z 6 6 675.95 N.D 26 D-BennylL-Benzyl Z 6 6 675.95 677.30**

[0163] Mass spectrometric analysis: The discrepancy between thecalculated and the observed mass as described in Table 1 ranges between1.5 to 2.5 amu. These results may indicate the existence of reduced noncyclic molecule rather then the oxidized desired macrocycles. In orderto negate this possibility the peaks were analyzed by splitting (markedwith ** in Table 1). This analysis yielded the expected MW values with adiscrepancy of only 0.3 amu. Furthermore, these molecules were alsoanalyzed by MS-MS and a fragment indicating a disulfide bridge wasfound:

1. Scaffold-based compound having the following general formula (B):

including pharmaceutically acceptable salts, esters or solvates thereof,wherein Z is selected from C═O and —CH₂—, W is selected from C═O and abond, provided that at least one of Z and W is C═O, X and Y areindependently selected from CH₂, O, S, NH, N—R⁵, NH—CO, CO, CH₂CO, S═Oand SO₂, or X and Y may form together a group selected from CH═CH,CO—NR⁵, NH—CO—N, O—CH(R⁵)—O, NH—CH(R⁵)—O and NH—CH(R⁵)—NH, where thehydrogen in the above groups may optionally be substituted by an alkylgroup, (a) and (b) are parts of the scaffold and are nitrogen containingbivalent organic radicals, each independently providing between 1 to 4atoms to said scaffold, R¹, R², R³ and R⁴ are each independentlyselected from H and substituted or unsubstituted allyl, and n and m areintegers from 1 to 6, with the exclusion of the following compound:glycinamide,L-tyrosyl-N-[2-[(2S)-4-[(1S)-1-carboxy-3-methylbutyl]-3,4,5,8-tetrahydro-3-oxo-2-(phenymethyl)-1,4-diazocin-1(2H)-yl]2-oxoethyl.2. A compound of the formula (B) according to claim 1, wherein X and Yare independently selected from CH₂, O, S, NH, N—R⁵, NH—CO, CO, CH₂CO,S═O and SO₂, or X and Y may form together a group selected from. CO—NR⁵,NH—CO—NH, O—CH(R⁵)—O, NH—CH(R⁵)—O and NH—CH(R⁵)—NH, where the hydrogenin the above groups may optionally be substituted by an alkyl group. 3.A compound according to claim 1, with each of parts (a) and (b)providing up to two atoms to said scaffold.
 4. A compound according toclaim 1, wherein each of parts (a) and (b) is independently selectedfrom —N(CHR⁶COL)—, —C(COL)(R⁶)—, —N(COR⁸)—CH⁷— and —C(NHR⁸)(R⁷)—. 5.Compound of the formula I, II, III or IV:

including pharmaceutically acceptable salts, esters or solvates thereof,wherein X and Y are independently selected from CH₂, O, S, NH, N—R⁵,NH—CO, CO, CH₂CO, S═O and SO₂, or X and Y may form together a groupselected from CH═CH, CO—NR⁵, NH—CO—NH, O—CH(R⁵)—O, NH—CH(R⁵)—O andNH—CH(R⁵)—NH, where the hydrogen in the above groups may optionally besubstituted by an alkyl group; R¹, R², R³ and R⁴ are each independentlyselected from H and substituted or unsubstituted alkyl; R⁵, R⁶, R⁷ andR⁸ are each independently selected from H, substituted or unsubstitutedalkyl, cycloalkyl, aryl, aralkyl, hetroaryl, heterocyclyl, hetroaralkyl,acyl, carboxyaryl, carboxyalkyl, side chains of naturally andartificially occurring amino acids as well as derivatives and mimics ofsuch side chains, and linear or cyclic peptide; L is selected from H,OH, NH₂, NHR⁵, a peptide and a solid support, where R⁵ is as definedabove, and n and m are integers from 1 to 6, with the exclusion of thefollowing compound: glycinamide,L-tyrosyl-N-[2-[(2S)-4-[(1S)-1-carboxy-3-methylbutyl]-3,4,5,8-tetrahydro-3-oxo-2-(phenymethyl)-1,4-diazocin-1(2H)-yl]2-oxoethyl.6. A compound of the formula I, II, III or IV according to claim 5,wherein X and Y may form together a group selected from CO—NR⁵,NH—CO—NH, O—CH(R⁵)—O, NH—CH(R⁵)—O and NH—CH(R⁵)—NH where the hydrogen inthe above groups may be substituted by an alkyl group.
 7. A compound ofthe formula I, II, III or IV according to claim 5, wherein X and Y formcombinations selected from: both X and Y are S; X is NH and Y is CO; Xis CO and Y is NH; X is NH and Y is CH₂; X is CH₂ and Y is NH; X and Yare NH—CO—NH; X is N—R⁴ and Y is CO; and X is CO and Y is N—R⁴.
 8. Acompound of the formula I according to claim
 5. 9. A compound of theformula II according to claim
 5. 10. A compound of the formula IIIaccording to claim
 5. 11. A compound of the formula IV according toclaim
 5. 12. A combinatorial library comprising a plurality of compoundsof the formula (B) as defined in claim
 1. 13. A combinatorial librarycomprising a plurality of compounds of any one of the formulae I, II,III or IV as defined in claim
 5. 14. A library according to claim 13,wherein in the compounds of the formulae I, II, III or IV, X and Y mayform together a group selected from CO—NR⁵, NH—CO—NH, O—CH(R⁵)—O,NH—CH(R⁵)—O and NH—CH(R⁵)—NH, where the hydrogen in the above groups maybe substituted by an alkyl group.
 15. A library according to claim 13,wherein in tHe compounds of formulae I, II, III and IV, the followingcombinations for X and Y are selected from the group consisting of: (i)X and Y are both S; (ii) X is NH and Y is CO; (iii) X is CO and Y is NH;(iv) X is N—R⁴ and Y is CO; and (v) X is CO and Y is N—R⁴.
 16. Apharmaceutical composition comprising a pharmaceutically acceptablecarrier and an effective amount of a compound of the formula I, II, IIIor IV as defined in claim
 5. 17. The composition of claim 16 for thetreatment of a disease, disorder or condition wherein a therapeuticallybeneficial effect is associated with the modulation of a protein orpeptide-mediated cell activity.
 18. A composition according to claim 16for use in veterinary.
 19. A composition according to claim 16 for usein human mammals.
 20. A compound of formula I, II, III or IV as definedin claim 5 for use in agriculture.
 21. Use of a compound of the formulaI, II, III or IV as defined in claim 5, for the preparation of apharmaceutical composition.
 22. A method for modulating protein orpeptide-mediated cell activity comprising contacting a cell componenthaving said protein or peptide with an effective amount of a compound offormula I, II, III or IV as defined in claim
 5. 23. A method for Hetreatment of a disease, disorder or condition wherein a therapeuticallybeneficial effect is associated with the modulation of a protein orpeptide-mediated cell activity, the method comprising administering to asubject in need of such treatment a therapeutically effective amount ofa compound of formula I, II, III or IV as defined in claim
 5. 24. Amethod according to claim 23 wherein said cell activity is selected fromproliferation, differentiation, cellular shape alteration, cellularelongation, uptake of substances by cells secretion of substances,cellular metabolism, and expression of various proteins.
 25. A method ofidentifying a candidate which modulates a protein or peptide-mediatedcell activity, such method comprising: (a) identifying in said proteinor peptide, a domain which is essential for said activity, (b)identifying in said domain, pharmacophors essential for the activity,(c) planning a combinatorial library of compounds having the formula I,II, III or IV as defined in claim 5, wherein each such compoundcomprises at least two of the pharmacophors identified in step (b) aboveor mimics or derivatives of the pharmacophors, where each member of thelibrary differs from the other by at least one of the following: i) thesize of the ring; ii) the order in which the pharmacophors are arrangedin the ring; iii) the chemical nature of the ring; iv) the chemicalnature of the pharmacophors; v) the chirality of the linker between thering and the pharmacophor; and vi) the chirality of the pharmacophor;(d) synthesizing a plurality of compounds among the compounds of thecombinatorial library planned in step (c); (e) screening the compoundssynthesized in step (d) for candidates that modulate said activity. 26.A method according to claim 25, comprising the following step after step(c) and before step (d): (c1) virtually screening on a computer thecompounds planned in step (b) to identify compounds with desirable 3Dstructures and selecting these compounds, and wherein step (d)comprises: synthesizing the selected compound of step (c1).
 27. A methodaccording to claim 25 wherein said activity is selected from:proliferation, differentiation, cellular shape alteration, cellularelongation, uptake of substances by cells, secretion of substances,cellular metabolism, and level of expression of various proteins.
 28. Amethod for obtaining compounds which modulate protein orpeptide-mediated cell activity, the method comprising: (1) identifyingthe candidate compounds according to the method of claim 25; (2)contacting the compounds identified in step (1) with a test assay fordetermining a protein or peptide-mediated cell activity; (3) collectingthose molecules which modulate a protein or peptide-mediated cellactivity in a test assay as compared to the modulation in the same testassay in the absence of said compound, thereby obtaining modulators ofsaid activity; and (4) producing the compounds obtained in step (3). 29.A method according to claim 28, wherein said activity is selected fromproliferation, differentiation, cellular shape alteration, cellularelongation, uptake of substances by cells, secretion of substances,cellular metabolism, and expression of various proteins.
 30. Modulatorof a protein or peptide-mediated cell activity obtained by the method ofclaim
 28. 31. A method for modulating a protein or peptide-mediated cellactivity, such method comprising contacting a cell whose activity is tobe modulated with a modulator obtained by the method of claim
 28. 32. Acompound of formula I, II, III or IV as defined in claim 5, being linkedeither directly or through a linker to a marker for imaging or to adrug.
 33. A compound according to claim 32, linked to a marker forimaging.
 34. A compound according to claim 33 wherein the marker is forthe detection of fluorescence, X-ray, MRI or radio-isotope scan.